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Biomedical subjects

A Vincent

Publications and source records attributed to A Vincent.

At least 19 recordsLinked to original sources
PubMed2000

Differential expression and subcellular localization of murine BRCA1 and BRCA1-delta 11 isoforms in murine and human cell lines.

BRCA1 mutations are involved in breast and ovarian cancer predisposition in humans. The biological functions of the murine BRCA1 gene have been extensively studied but little is known about murine BRCA1 proteins. To better characterize these proteins, we have cloned the full-length murine BRCA1 cDNA and a splice variant deleted of exon 11, BRCA1-delta 11, by RT-PCR method. Three polyclonal antibodies raised against various parts of murine BRCA1 were used in our study: D16, M20 and 5MO, which were generated in our laboratory. This allowed us to analyze the expression and subcellular localization of both isoforms in murine and human cell lines by immunoblotting, immunoprecipitation, cell fractionation and immunofluorescence. Endogenous BRCA1 was detected in murine cell lines but not splice variant BRCA1-delta 11, whereas both ectopically expressed murine isoforms were detected in transfected human Bosc 23 cells. Subcellular fractionation and immunofluorescence results showed that the BRCA1 protein was mainly located in the nucleus, whereas BRCA1-delta 11 was preferentially cytoplasmic. The conservation of exon 11 splicing and the differential subcellular localization of BRCA1 and BRCA1-delta 11 in human and mouse suggest that these proteins could play distinct roles and that they could differentially act in the pathological mechanisms leading to the development of breast and ovarian cancer. The characterization of the murine BRCA1 proteins and antibodies will be useful to further study BRCA1 functions in murine models.

3T3 Cells↗
PubMed2000

Group I and group III metabotropic glutamate receptor subtypes provide enhanced neuroprotection.

Neuroprotection by the metabotropic glutamate receptor (mGluR) system has been linked to the modulation of both the free radical nitric oxide (NO) and programmed cell death (PCD). Because the cellular mechanisms that ultimately determine neuronal PCD rely upon the independent pathways of genomic DNA degradation, externalization of membrane phosphatidylserine (PS) residues, and the activation of associated cysteine proteases, we investigated the ability of the individual mGluR subtypes to modulate the distinct pathways of NO-induced PCD in primary rat hippocampal neurons. Membrane PS residue externalization occurred within the initial 3 hr after exposure to the NO donors (300 microM SNP or 300 microM NOC-9), preceded genomic DNA fragmentation, and was present in 80 +/- 2% of the neurons within a 24-hr period. NO exposure also led to the rapid induction of both caspase 1-like and caspase 3-like activities that were determined to be necessary, at least in part, for the generation of NO-induced genomic DNA degradation, but distinct from the detrimental effects of intracellular acidification. Yet, only caspase 1-like activity was necessary for the modulation of PS residue externalization. Activation of group I mGluR subtypes utilized an effective, "upstream" mechanism for the inhibition of cysteine protease activity that offered an enhanced level of neuroprotection through both the preservation of genomic DNA integrity and the maintenance of PS membrane asymmetry. Group II and Group III mGluR subtypes maintained DNA integrity and group III mGluR subtypes additionally prevented PS residue externalization through mechanisms that were targeted below the level of caspase activation. Our work elucidates the independent nature of the mGluR subtypes to not only provide discrete levels of protection against neuronal PCD, but also offer robust therapeutic strategies for neurodegenerative disease.

Animals↗
PubMed2000

Antisense suppression of potassium channel expression demonstrates its role in maturation of the action potential.

A developmental increase in delayed rectifier potassium current (I(Kv)) in embryonic Xenopus spinal neurons is critical for the maturation of excitability and action potential waveform. Identifying potassium channel genes that generate I(Kv) is essential to understanding the mechanisms by which they are controlled. Several Kv genes are upregulated during embryogenesis in parallel with increases in I(Kv) and produce delayed rectifier current when heterologously expressed, indicating that they could encode channels underlying this current. We used antisense (AS) cRNA to test the contribution of xKv3.1 to the maturation of I(Kv), because xKv3.1 AS appears to suppress specifically heterologous expression of potassium current by xKv3.1 mRNA. The injection of xKv3.1 AS into embryos reduces endogenous levels of xKv3.1 mRNA in the developing spinal cord and reduces the amplitude and rate of activation of I(Kv) in 40% of cultured neurons, similar to the percentage of neurons in which endogenous xKv3.1 transcripts are detected. The current in these mature neurons resembles that at an earlier stage of differentiation before the appearance of xKv3.1 mRNA. Furthermore, AS expression increases the duration of the action potential in 40% of the neurons. No change in voltage-dependent calcium current is observed, suggesting that the decrease in I(Kv) is sufficient to account for lengthening of the action potential. Computer-simulated action potentials incorporating observed reductions in amplitude and rate of activation of I(Kv) exhibit an increase in duration similar to that observed experimentally. Thus xKv3.1 contributes to the maturation of I(Kv) in a substantial percentage of these developing spinal neurons.

Action Potentials↗
PubMed2000

Autoantibody screening in subacute cerebellar ataxia.

In a retrospective study of 280 sera from patients presenting with cerebellar signs, seven of whom had proved positive for the typical paraneoplastic serum antibodies that were requested by the clinicians, raised concentrations of antibodies to voltage-gated calcium channels or to glutamic acid decarboxylase were detected in a further seven sera. Systematic screening for these and other antibodies in future cases should help in the diagnosis and management of the patients.

Autoantibodies↗
PubMed2000

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis.

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using 'top-down' elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.

Adenosine Triphosphate↗
PubMed2000

Asymptomatic maternal myasthenia as a cause of the Pena-Shokeir phenotype.

We report six sibs with arthrogryposis multiplex congenita and a Pena-Shokeir phenotype, born to a healthy woman who was discovered to have asymptomatic myasthenia gravis (MG). This is the first report of anti-acetylcholine receptor (AChR) antibodies causing fetal akinesia/hypokinesia sequence in the offspring of an asymptomatic mother.

Abnormalities, Multiple↗
PubMed2000

First evidence of uncoupling protein-2 (UCP-2) and -3 (UCP-3) gene expression in piglet skeletal muscle and adipose tissue.

Uncoupling proteins (UCPs) facilitate proton transport inside the mitochondria and decrease the proton gradient, leading to heat production. Until now, the presence of UCP1 or other UCP homologs had not been detected in tissues of pig, a species where evidence for the presence of brown adipose tissue has only been provided in 2-3 month old animals. In the light of the improving knowledge on the UCPs family, we decided to examine both UCP2 and UCP3 mRNA expression in piglet skeletal muscle and adipose tissue. Using RT-PCR we have successfully cloned a partial UCP2 sequence and a complete UCP3 cDNA. UCP3's open reading frame (936bp) shares 90, 89 and 85% similarity with bovine, human and rat UCP3 nucleotide sequences, respectively. In 3-5 day old piglets, these genes are expressed in adipose tissue and in both longissimus thoracis (LT) and rhomboïdeus (RH) muscles, without any effect of muscle metabolic type. This is in good agreement with the measurement of the same membrane potential in mitochondria isolated from both types of muscles. In triiodothyronine-treated piglets, UCP3 mRNA is more expressed in LT than in RH muscle. These genes may be involved in the control of the energy metabolism of the piglet.

Adipose Tissue↗
PubMed2000

Autoantibodies in thymoma-associated myasthenia gravis with myositis or neuromyotonia.

BACKGROUND: About 50% of patients with thymoma have paraneoplastic myasthenia gravis (MG). Myositis and myocarditis or neuromyotonia (NMT) will also develop in some. Patients with thymoma-associated MG produce autoantibodies to a variety of neuromuscular antigens, particularly acetylcholine receptor (AChR), titin, skeletal muscle calcium release channel (ryanodine receptor [RyR]), and voltage-gated potassium channels (VGKC). OBJECTIVE: To examine whether neuromuscular autoantibodies in patients with thymoma correlate with specific clinical syndromes. METHODS: Serum and plasma samples from 19 patients with thymoma-associated MG, of whom 5 had myositis and 6 had NMT, underwent testing for antibodies to AChR, titin, RyR, and VGKC. RESULTS: Antibodies to AChR and titin were found in 19 and 17 patients, respectively. Antibodies to RyR correlated with the presence of myositis (P = .03); they were found in all 5 patients with myositis and in only 1 patient with NMT, but also in 4 of 8 patients with neither disease. Antibodies to VGKC were found in 4 patients with NMT, 1 of 3 patients undergoing testing for myositis, and 2 of 7 patients undergoing testing with neither comorbidity. Presence of RyR antibodies correlated with high levels of titin antibodies. CONCLUSIONS: The results appear to distinguish partially between 3 groups of patients with thymoma-associated MG: the first with RyR antibodies and myositis or myocarditis, the second with NMT without RyR antibodies, and the third without RyR antibodies, myositis, or NMT. Differences in the thymoma may underlie these pathologic associations.

Adult↗
PubMed2000

Teratogen update: maternal myasthenia gravis as a cause of congenital arthrogryposis.

BACKGROUND: Arthrogryposis multiplex congenita (AMC) is defined as nonprogressive congenital contractures that generally result from lack of fetal movement in utero. AMC is a feature of many congenital disorders caused by genetic, environmental, or other factors. One rare cause of AMC is maternal myasthenia gravis (MG). This is an autoimmune disorder, caused by antibodies to the nicotinic acetylcholine receptor (AChR), and resulting in weakness of voluntary muscles. In 10-15% of babies born to MG mothers, transient signs of MG are noted after placental transfer of anti-AChR antibodies. In a few cases, AMC predominates. METHODS: We review the role of antibodies to AChR in MG and in AMC associated with maternal antibodies to AChR. RESULTS: In anti-AChR antibody-associated AMC, fetal or neonatal death is common; other deformities or CNS abnormalities are common as well. The condition usually recurs in each pregnancy unless the mother is treated for MG, but some mothers are asymptomatic. The maternal antibodies cross the placenta and block the function of the fetal isoform of the AChR leading to fetal paralysis. Injection of maternal plasma into pregnant mice results in AMC in mouse fetuses. Some women with recurrent AMC in their babies have no detectable anti-AChR suggesting the presence of antibodies to other fetal muscle or neuronal proteins. CONCLUSIONS: Although rare, anti-AChR-associated AMC is potentially treatable and can be diagnosed by a routine antibody test. The mouse model can be used to investigate the role of these and other maternal antibodies in causing congenital conditions.

Animals↗
PubMed2000

Dramatic inhibition of amiodarone metabolism induced by grapefruit juice.

AIMS: Grapefruit juice increases blood concentrations of many drugs metabolized by CYP3A. Amiodarone is metabolized by CYP3A to N-desethylamiodarone (N-DEA). The aim of this study was to determine amiodarone kinetics when administrated with and without grapefruit juice. METHODS: Eleven healthy adult volunteers took part in a single sequence, repeated-measures design study. Each subject, who had been evaluated 6 months previously for amiodarone pharmacokinetics, was given a single oral dose of amiodarone (17 mg kg-1) with three glasses of 300 ml of grapefruit juice on the same day. RESULTS: Grapefruit juice completely inhibited the production of N-DEA, the major metabolite of amiodarone, in all subjects and increased the area-under-the-curve (AUC) and maximum concentration of amiodarone (Cmax) by 50% and 84%, respectively, as compared with the control period during which water had been administrated instead of grapefruit juice (AUC: 35.9 +/- 14.3 vs 23.9 +/- 11.2 microg ml-1 h, P < 0.005 and Cmax: 3.45 +/- 1.7 vs 1.87 +/- 0.6 microg ml-1, P < 0. 02, respectively) (means +/- s.d.). This inhibition of N-DEA production led to a decrease in the alterations caused by amiodarone on PR and QTc intervals. CONCLUSIONS: Grapefruit juice dramatically alters the metabolism of amiodarone with complete inhibition of N-DEA production. These results are in agreement with in vitro data pointing to the involvement of CYP3 A in the metabolism of amiodarone and suggests that this interaction should be taken into account when prescribing this antiarrhythmic drug.

Adult↗
PubMed2000

Molecular targets for autoimmune and genetic disorders of neuromuscular transmission.

The neuromuscular junction is the target of a variety of autoimmune, neurotoxic and genetic disorders, most of which result in muscle weakness. Most of the diseases, and many neurotoxins, target the ion channels that are essential for neuromuscular transmission. Myasthenia gravis is an acquired autoimmune disease caused in the majority of patients by antibodies to the acetylcholine receptor, a ligand-gated ion channel. The antibodies lead to loss of acetylcholine receptor, reduced efficiency of neuromuscular transmission and muscle weakness and fatigue. Placental transfer of these antibodies in women with myasthenia can cause fetal or neonatal weakness and occasionally severe deformities. Lambert Eaton myasthenic syndrome and acquired neuromyotonia are caused by antibodies to voltage-gated calcium or potassium channels, respectively. In the rare acquired neuromyotonia, reduced repolarization of the nerve terminal leads to spontaneous and repetitive muscle activity. In each of these disorders, the antibodies are detected by immunoprecipitation of the relevant ion channel labelled with radioactive neurotoxins. Genetic disorders of neuromuscular transmission are due mainly to mutations in the genes for the acetylcholine receptor. These conditions show recessive or dominant inheritance and result in either loss of receptors or altered kinetics of acetylcholine receptor channel properties. Study of these conditions has greatly increased our understanding of synaptic function and of disease aetiology.

Binding Sites↗
PubMed2000

Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programmed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 micromol/L) alone rapidly increased genomic DNA fragmentation from 20 +/- 4% to 71 +/- 5% and membrane PS exposure from 14 +/- 3% to 76 +/- 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.

Animals↗